Compounds based on an S-acyl-2-mercaptobenzamide thioester (SAMT) template have been shown to specifically eject zinc from the C-terminal zinc-binding domain (ZD2) of NCp7 via acyl transfer from the thioester to the sulfur of a zinc-coordinating cysteine residue. These zinc-binding domains are excellent targets for the development of new antiretroviral and microbicidal agents because of their structural conservation and the broad range of functions of NCp7 in the viral replication cycle. Our previous work identified several lead SAMT compounds that were shown to be virucidal and to inhibit cell-to-cell transmission of HIV-1 in co-culture systems. In addition, based on mutational analysis of the NCp7 amino acid sequence, we were able to extend the previously reported mechanism of action of the SAMT compounds to include a secondary S to N intramolecular acyl transfer that occurs after the primary transfer from the thioester to a cysteine side chain in the protein. This essential step is irreversible and results in permanent disruption of the functional native structure. We have finally identified that the same covalent modifications take place in cells treated with the SAMT compounds, giving rise to aggregated protein that is unable to be cleaved by protease for viral maturation. We are currently evaluating the SAMT compounds for use as vaginal, topical microbicides. As part of these efforts, we are exploring ways to formulate the SAMT compounds for sustained delivery. In collaboration with Thomas Smith at the Oak Crest Institute of Science, we have developed a silicone vaginal ring that incorporates one of our lead SAMT compounds. We observed linear release of the SAMT from the ring in vitro, and we will be proceeding with safety and efficacy studies of the SAMT in a non-human primate model. The vaginal ring developed by Dr. Smith allows the incorporation of different antiviral agents in the same ring. We have examined the effects of combining SAMT compounds with other HIV inhibitors, including tenofovir, in collaboration with Dr. Robin Shattock of St. Georges University of London. We found that the SAMTs act additively to synergistically with all compounds tested. We plan to build on these results by examining the safety and efficacy of vaginal rings containing both a lead SAMT and a second antiretroviral compound in non-human primates. We are also using mass spectrometry experiments to elucidate the metabolism and pharmacokinetics of the SAMT compounds. In collaboration with investigators at Waters Corporation, we have examined the metabolites of the SAMT compounds following short-term culture with vaginal biopsy tissue from pig-tailed macaques. The primary compounds identified were the thioester itself, the thiol released by reaction with NCp7 or hydrolysis, and a benzisothiazolone that results from cyclization of the thiol. We will continue to investigate the activity and metabolism of the lead SAMT compounds in cellular model systems. Identification of a potentially safe and efficacious single or combination candidate microbicide in non-human primates, and the elucidation of their pharmacokinetics, will lead the way into studies necessary for preclinical evaluation of the SAMT compounds. While conducting our studies on the mechanism of viral inactivation in cells, we observed that the thiol generated by reaction of the SAMT with NCp7 had similar antiviral activity as the SAMT itself. This result was surprising, as the thiol was not active in vitro. We observed that the thiol induced the same aggregation of unprocessed protein as treatment with the thioester itself. Moreover, we found that thiol treatment resulted in radiolabeling of the Gag precursor of NCp7 in the presence of 14C-labeled acetate or 14C-labeled pyruvate, which is converted to acetate in the cell. This activity was specific to the thiol form of the SAMTs, as movement of the thiol bond from the ortho to the para position abrogated all activity against Gag. Thus, we proposed that the thiol is re-acylated intracellularly by reaction with acetyl CoA to form an active thioester capable of modifying the NCp7 domain of Gag. The observation of the recycling mechanism led us to design a prodrug derivative in which a para-hydroxybenzyl ester was used to mask the sulfur atom. Inside the cell, the oxygen ester of the thioether prodrug can be cleaved by esterases to yield an intermediate that breaks down via 1,4-elimination, releasing the thiol for acetylation to form the antiviral thioester. As expected, we found that the thioether prodrug did not react with NCp7 in vitro, but in cytoprotection assays using cultured and primary cells, it had antiviral activity similar to the SAMT. Moreover, virions from cells treated with the thioether prodrug and 14C-labeled pyruvate showed radiolabeling of Gag similar to the SAMT and thiol. Thus, treatment with the thioether prodrug produced results similar to those of the SAMTs in virions from infected cells, confirming our hypothetical mechanism of action that the thiol produced by reaction with NCp7 can be recycled intracellularly to form an active thioester. Our research has demonstrated a novel mechanism by which the SAMT compounds can be repeatedly regenerated intracellularly. This mechanism is critical to the antiviral activity of the SAMTs and is unique to this class of antiviral inhibitors. Thus, our research demonstrates the viability of the SAMTs as a candidate microbicide to prevent the transmission of HIV and we will continue to explore their use in the future.